Study Of Regulative Mechanisms Of Cid/Lrg Regulatory System To The Biofilm Formation Of Staphylococcus Epidermidis Isolation

Staphylococcus epidermidis is a species of opportunistic pathogens. Its pathogenicity is associated with biofilm formation. Some studies have confirmed that cell death and lysis controlled by cid and lrg and the extracellular DNA(eDNA) releasing are important for the biofilm formation and stabilization. Intervention of bacterial cell death and lysis may be a new way to control the biofilm. This study focus on regulation of S. epidermidis biofilm formation, analyze the influence of different eDNA levels on the ability of biofilm formation of S. epidermidis isolates. Observe the influence of glucose,100μM CCCP and Omeprazole on biofilm formation to supply a basis for the clinical therapies of biofilm-related infections.Objective:①To investigate the relationships between the expressions of cidA and lrgA and their regulative genes and the biofilm formation of S. epidermidis clinical isolates.②To investigate the influence of different eDNA levels in S. epidermidis isolates on the ability of biofilm formation.③To investigate the influence of glucose, efflux pump inhibitors CCCP and Omeprazole on S. epidermidis biofilm and the possible mechanism among them.Methods:①227S. epidermidis clinical isolates were divided into biofilm formation(BF) group and non-biofilm formation(NBF) group.②Amplify cidA and lrgA fragment with PCR, detect mRNA levels of cidA and lrgA with RT-PCR and then calculate the ratios of cidllrg mRNA.③Amplify cidR and lytSR fragment with PCR and contrast DNA sequense with RP62A and ATCC12228.④Detect eDNA levels of S. epidermidis in planktonic culture and microtitre plate static culture.⑤The eDNA in S. epidermidis biofilms stained by AO-PI was observed by fluorescence microscopy and by CLSM.⑥Detect the cidA and IrgA mRNA levels, eDNA levels and biofilm-forming ability of S. epidermidis with RT-PCR, microtitre plate culture and adhesion assays when adding35mM glucose,100μM CCCP and25,50,100,200μg/ml Omeprazole. Results:①26isolates were positive in adhesion assays and32isolates existed icaA gene among227S. epidermidis isolates. Select20isolates of BF group and19isolates of NBF group for the later experiments.②All of the39isolates existed cidA and lrgA gene. The relative levels of cidA mRNA were0.340±0.250in BF group and0.406±0.408 (P=0.541) in NBF group for4h’s culture, while the relative expression levels of lrgA were0.325±0.218and0.253±0.211respectively (P=0.299). There were no significant differeces between these two groups.③The ratios of cid/lrg mRNA were1.067±0.529and1.958±1.877respectively, the difference of population distribution was significant (P=0.001).④The highest level of cidA mRNA of Y36and Y26shows on4h’s culture, while lrgA mRNA shows on6h.⑤All of the39isolates existed cidR and lytSR gene.⑥The eDNA levels were32.2±10.1(μg/ml),33.6±11.9(μg/ml),34.3±10.0(μg/ml) in20BF isolates when cultured in planktonic condition for2,4,6h, while in19NBF isolates were28.7±8.9(μg/ml),31.5±11.7(μg/ml),31.8±12.7(μg/ml) respectively. There was no significant differeces between the two groups for these three phases(P>0.05). The eDNA levels were740.0±264.4(ng/OD600) in20BF isolates when cultured in static microtitre plate, higher than that of19NBF isolates,80.1±31.1(ng/OD600), and the difference between these two groups was significant.⑦The eDNA in BF isolate Y36biofilms could be visualized by staining with AO and PI, while neither biofilm structure nor eDNA appeard when NBF isolate Y26was cultured for24h.⑧When35mM glucose was added, cidA and lrgA mRNA levels increased, eDNA levels and adherence increased.⑨When100μM CCCP or Omeprazole at a certain concentration range was added, lrgA mRNA levels increased and eDNA levels decreased, adherence also decreased. Conclusion:①The expressions of cidA and lrgA of S. epidermidis clinical isolates may be time-limited. The ratios of cidA/lrgAmRNA maybe have some biological significance.②eDNA is one of the important matrix components in the S. epidermidis biofilm-forming process. The eDNA of static culture in microtitre plate was more efficient than planktonic culture in the case of estimating the ability of biofilm formation of S. epidermidis isolates.③35mM glucose and100μM CCCP can change cidA/lrgA system transcription and eDNA releasing, and then influence bacterial adherence. Omeprazole, to some extent, can increase lrgA transcription, decrease eDNA releasing, and thus inhibit the biofilm formation of S. epidermidis.